DNA binding proteins bind to DNA in both sequence specific and nonspecific modes in vivo and in vitro. The extent of gene expression is controlled by the extent to which the protein overcomes the competing nonspecific DNA and binds to its specific site. Various p[physiologically relevant factors may affect the ratio of specific to nonspecific binding. This project is attempting to determine the role of hydration and water release in the stability and specificity of recognition. When the two macromolecules bind, some of the interactions with the milieu are lost, and new interactions with the complementary surface are formed. This exchange of interactions with water and ions for interactions with the other macromolecule makes the extent of binding very sensitive to solution conditions. In addition, DNA-mediated protein- protein interactions (e.g. DNA looping), are an important part of the biological activity of most transcriptional regulators. By quantitating the binding in the presence of high concentrations of small molecule solutes, one can determine the effect of surface hydration of both protein and DNA on complex formation, and therefore the total number of water molecules released in the binding reaction, which should be related to the amount of surface area of both protein and DNA occluded in the complex. Such insight into the hydration of both surfaces before and after complexation is important for understanding the sources of stability and specificity of a protein-DNA complex. We have shown that this water release phenomenon occurs only for specific binding, which suggests a mechanism for gene regulation in response to high osmotic stress.